Antifungal drug resistance evoked via RNAi-dependent epimutations

Abstract

Microorganisms evolve via mechanisms spanning sexual/parasexual reproduction, mutators, aneuploidy, Hsp90, and even prions. Mechanisms that may seem detrimental can be repurposed to generate diversity. Here we show the human fungal pathogen Mucor circinelloides develops spontaneous resistance to the antifungal drug FK506 (tacrolimus) via two distinct mechanisms. One involves Mendelian mutations that confer stable drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable drug resistance. The peptidyl-prolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin1. Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth2. Mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance (FK506R) and restore hyphal growth. In parallel, RNAi is spontaneously triggered to silence the FKBP12 fkbA gene, giving rise to drug-resistant epimutants. FK506R epimutants readily reverted to the drug-sensitive wild-type (WT) phenotype when grown without drug. The establishment of these epimutants is accompanied by generation of abundant fkbA small RNA (sRNA) and requires the RNAi pathway as well as other factors that constrain or reverse the epimutant state. Silencing involves generation of a double-stranded RNA (dsRNA) trigger intermediate from the fkbA mature mRNA to produce antisense fkbA RNA. This study uncovers a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes.

A non-canonical RNA degradation pathway suppresses RNAi-dependent epimutations in the human fungal pathogen Mucor circinelloides

“…In fact, two novel and unusual mechanisms have been recently reported in the human fungal pathogen Mucor circinelloides that differ from the canonical exogenously or endogenously activated RNAi pathways described previously [6, 7]. These are an RNAi-dependent epimutation pathway [4] and an RdRP-dependent Dicer-independent degradation cascade that acts on endogenous mRNA [5]. These discoveries position M .…”

“…Quelling, meiotic silencing by unpaired DNA (MSUD), and sex induced silencing (SIS) are distinct RNAi pathways that have been described in Neurospora crassa and Cryptococcus neoformans , respectively, and new RNAi pathways are still being discovered in other fungi [4, 5]. In fact, two novel and unusual mechanisms have been recently reported in the human fungal pathogen Mucor circinelloides that differ from the canonical exogenously or endogenously activated RNAi pathways described previously [6, 7].…”

Glycan Alteration Imparts Cellular Resistance to a Membrane-Lytic Anticancer Peptide

“…In general, resistance towards toxic agents is acquired through genetic mutations, amplification of a particular gene, or change of gene expression caused by epigenetic alterations (Calo et al, 2014). To test for these possibilities, we analyzed the survival of colonies originating from random spores of the putative pvg2-1 mutant.…”

Epigenetic Control of Phenotypic Plasticity in the Filamentous Fungus Neurospora crassa

“…The canonical RNA interference pathway is not required for DNA methylation (Freitag et al 2004), and it is not known if RNA-directed epigenetic modifications, such as the plant RNA-directed methylation pathway (Matzke and Mosher 2014), exist or if RNA molecules can mediate epigenetic inheritance like in animals (Rassoulzadegan et al 2006; Rechavi et al 2011; Ashe et al 2012) or other fungi (Qutob et al 2013; Calo et al 2014). However, a class of small RNAs called disiRNAs, may be involved in controlling DNA methylation at specific loci (Dang et al 2013).…”